Isolation and Characterization of

Non-Saponifiable and Saponifiable

Lipids from Calfs Brain
Abella, Angelica
Balmes, Angela

- 3B8 Bustillo, John Paul

Dalupang, Audrey
Figueroa, Cyreen

Introduction

What is a
Lipid?
a heterogeneous class of naturally
occurring organic compounds
classified together on the basis of
common solubility properties.

hydrophobic
(insoluble in water
=nonpolar) , but
soluble in aprotic
organic solvents
Amphipathic in
nature

Functions:
1. Cell membrane component
a. Phospholipids (Glycerophospholipids and
Sphingolipids)
b. Glycolipids (Sphingolipids)
2. Energy storage and Insulation
Triacylglycerol
3. Signaling molecules

Calfs Brain (Beef brain)

Brain tissues contains high amount of lipid content
Contains complex lipids such as phosphatides, cerebrosides,
sphingosines and cholesterol
It also contain 17% grams of fat and 4% of saturated fat
Brain tissues are rich in saponifiable and non-saponifiable
lipids which are important to life processes.

Saponifiable Lipid
Simple Lipids-fatty acid ester of different alcohols and carries no
other substance (Example: fatty acids and triglycerides)
Complex Lipids-esters of fatty acids with alcohol and contain
additional groups
Phosphorylated- containing a phosphoester group
(Example: glycerophosphatides, sphingosine
phosphatides)

Non-phosphorylated- do not contain phosphoester

group (Example: Sphingosine glycosides)

Non-saponifiable Lipid
Does not contain ester linkages
Not hydrolyzed by sodium hydroxide or other basic compounds
Example: cholesterol and prostaglandins

Gravity Filtration
Method used either for the removal of solid
impurities from an organic solution or for isolation
of organic solid through the use of filter paper and
funnel
Hot Gravity Filtration

Used to separate insoluble impurities from a hot

solution

Prevents the crystals to form in the filter funnel

which then blocks the filtration process

Cholesterol
- A type of sterol (modified
steroid) which serves as the
biosynthetic precursor needed
to synthesize steroid hormones,
bile acids, and vitamin D.
- Synthesized by all animals
- Lack of ester functional group
makes it non-saponifiable

Glycerophospholipid - A type of lipid wherein two fatty

acids are attached by an ester

linkage to the 1st and 2nd
carbon of glycerol while a highly
polar/charged group is attached
to the 3rd carbon by a
phosphodiester linkage

- Forms the lipid bilayer of cell

membranes:
Phosphate group = polar head
Fatty acids = non-polar tails

- Most glycerophospholipids
are identified through X
head group attached to the
phosphate group

Sphingolipid
-

a class of lipids with a polar head

group and two nonpolar tails

The core of a sphingolipid is an

amino alcohol called sphingosine

Abundant in Central Nervous

System

They also serve as adhesion sites

for extracellular proteins and play
important roles in signal
transmission and cell recognition

Lecithin

- Also known as phosphatidylcholine

- It is a phospholipid which consists of glycerol, two
fatty acids, a phosphate group and choline.
- serves a structural role in cell membranes

Galactocerebroside
- A type of cerebroside
consisting of a ceramide* with
a galactose residue at the 1hydroxyl moiety
- It is also composed of
sphingosine and fatty acid
*ceramide: the amino group of
sphingosine is attached to the
fatty acid by an amide linkage

CHEMICAL
TESTS

Liebermann-Burchard Test
- Test for the presence of
Cholesterol
Salkowski Test
- Test for the presence of
Cholesterol
Phosphate Test
- Test for the presence of
Phosphate
Krauts Test
- Test for the presence of
Choline
Ninhydrin Test
- Test for the presence of free
amino groups and secondary
amines
Molisch Test
- Test for the presence of
Carbohydrates

Objectives

Objectives
To isolate lipids from calfs brain and to separate it into
phosphorylated and non-phosphorylated lipids
To characterize the isolated lipids and standards using
various chemical tests

Methodology

A.Isolation of Cholesterol
Calfs Brain - Part A

Filtrate (Cholesterol)
Evaporate using steam bath
Cool over ice bath
Filter and collect the crude
product
Dissolve in (1:3) MeOH:CHCl3

Residue - Part B

B. Isolation of Glycerophosphatides
Residue - Part B

Filtrate (Glycerophosphatides)
Concentrated over steam bath
Add 30mL acetone
Decant and dissolve the
precipitate in 10mL (1:3)
MeOH:CHCl3

Wash with acetone

Transfer to small beaker
Add 30mL hexane
Stir occasionally for 30 minutes
Filter

Residue - Part C

C. Isolation of Sphingosine Phosphatides

and Sphingosine Glycosides
Residue - Part C

Filtrate (discard)

Transfer to a small beaker

Add 50mL EtOH
Boil over water bath
Filter

Precipitate (Sphingosine)
Dissolve in 5mL (1:3)
MeOH:CHCl3

Characterization: Liebermann-Burchard Test

Characterization: Salkowski Test

20

Characterization: Test for Phosphate

-ignite over a free flame until all

organic matter is burned away

-heat 65 oC

Characterization: Krauts Test

Characterization: Ninhydrin Test

Characterization: Molisch Test

Results and Discussion

Isolation of Non-saponifiable from

Saponifiable Lipids from Calfs Brain
Trituration
- A process used to purify crude chemical compounds containing
soluble impurities.
Recrystallization
- Used to purify organic compounds
- The principle behind recrystallization is that the amount of solute
that can be dissolved by a solvent increases with temperature.

Isolation of Non-saponifiable from

Saponifiable Lipids from Calfs Brain
Acetone
- Used to separate the non-phosphorylated lipids from phosphorylated lipids
- Non-phosphorylated lipids are dissolved in acetone solution because they
are both polar
- Less reactive to the lipid components compared to alcohol
95% Hot EtOH
- Forms a concentrated solution
- Cholesterols solubility is directly proportional to the temperature
- Selective solvent for sphingolipids

Isolation of Non-saponifiable from

Saponifiable Lipids from Calfs Brain
Chloroform-Methanol solvent mixture (MeOH:CHCl3)
dissolves/ miscible with lipids. Moreover, polar lipids dissolve in methanol.
Chloroform = nonpolar solvent used in dissolving lipids
Methanol = polar component used for further isolation of remaining compounds in
the lipids
Hexane
good solvent only for lipids of low polarity
Its main use is to extract neutral lipids from mixtures of water with alcohols
With a fairly high volatility and a low sensible heat it is relatively easy to remove
from the solids and oil with low energy use.
The low boiling point of hexane (67C / 152F) and the high solubility of oils and
fats in it are the properties exploited in the solvent extraction process.
Used as a selective solvent of glycerophosphatides to separate them from
sphingolipids

Isolation of Non- Saponifiable and Saponifiable Lipids from Calfs Brain

Hexane Solvent Extraction

Solvent Extraction is a process which involves extracting oil fro

materials by treating it with a low boiler solvent as opposed to
by mechanical pressing methods (such as expellers, hydraulic p

This method recovers almost all the oils and leaves behind onl
residual oil in the raw material.

Because of the high percentage of recovered oil, solvent extrac

the most popular method of extraction of oils and fats.

Test for: Cholesterol

Reagents: Acetic anhydride and Sulfuric acid
Principle: Acetylation of the hydroxyl group of
cholesterol located at C-3 then reaction with
concentrated sulfuric acid
Positive Result: Emerald green solution

Liebermann-Burchard Test

A. Liebermann-Burchard Test
(+)Emerald green solution
STANDARDS
GROUP

GP

SP
C

LECITHIN

GLC

A. Liebermann-Burchard Test
(+)Emerald green solution
STANDARDS
GROUP

GP

SP
C

LECITHIN

10

GLC

Mechanism

POSITIVE: CHOLESTEROL & GALACTOCEREBROSIDE

Test for: Cholesterol

Reagents: concentrated Sulfuric acid
Principle: Addition of sulphuric acid reacts with
cholesterol resulting to red biocholestadien
disulfonate then dehydration forming a
bisteroid
Positive Result: red interphase

Salkowskis Test

B. Salkowskis Test
(+) Red interphase
STANDARDS
GROUP

GP

SP
C

LECITHIN

GLC

B. Salkowskis Test
(+) Red interphase
STANDARDS
GROUP

GP

SP
C

LECITHIN

10

GLC

Mechanism

POSITIVE: CHOLESTEROL & GALACTOCEREBROSIDE

Test for: Phosphate

Reagents: fusion mixture, 3M HNO3, 2.5% Ammonium
molybdate
Principle: Oxidation reaction, conversion of organic form
to inorganic phosphate; Precipitation reaction
Positive Result: yellow precipitate

Phosphate Test

C. Phosphate Test
(+) Yellow precipitate
STANDARDS
GROUP

GP

SP
C

LECITHIN

GLC

C. Phosphate Test
(+) Yellow precipitate
STANDARDS
GROUP

GP

SP
C

LECITHIN

10

GLC

Mechanism

POSITIVE: GLYCEROPHOSPHATIDE & LECITHIN

Test for: presence of choline

Reagents: Krauts reagent, Potassium iodide + Bismuth
subnitrate and Nitric acid
Principle: Complexation reaction of choline with bismuth
potassium iodide
Positive Result: dark orange or red precipitate

Krauts Test

D. Krauts Test
(+)Orange-red precipitate
STANDARDS
GROUP

GP

SP
C

LECITHIN

GLC

D. Krauts Test
(+)Orange-red precipitate
STANDARDS
GROUP

GP

SP
C

LECITHIN

GLC

10

Mechanism

POSITIVE: GLYCEROPHOSPHATIDE
SPHINGOSINE
LECITHIN

Test for: Free amino groups and secondary amines

Reagents: Ninhydrin
Principle: Oxidative deamination followed by
condensation
Positive Result: blue-violet solution

Ninhydrin Test

E. Ninhydrin Test
(+)Violet solution
STANDARDS
GROUP

GP

SP
C

LECITHIN

GLC

E. Ninhydrin Test
(+)Violet solution
STANDARDS
GROUP

GP

SP
C

LECITHIN

GLC

10

Mechanism

POSITIVE: GLYCEROPHOSPHATIDE, SPHINGOSINE & LECITHIN

Test for: Carbohydrates

Reagents: -naphthol in ethanol, concentrated Sulfuric
acid
Principle: Dehydration forming furfural and its derivative
Positive Result: violet interphase

Molisch Test

F. Molisch Test
(+) Violet interphase
STANDARDS
GROUP

GP

SP
C

LECITHIN

GLC

F. Molisch Test

(+) Violet interphase

STANDARDS

GROUP

GP

SP
C

LECITHIN

GLC

10

Mechanism

POSITIVE: GLYCEROPHOSPHATIDE, SPHINGOSINE, LECITHIN

GALACTOCEREBROSIDE

IDEAL RESULT

TESTS

Cholesterol

Glycerophos
phatide

Sphingosine

Lecithin

Galactocereb
roside

LiebermannBurchard
Test

Salkowski

Phosphate

N/A

N/A

N/A

Krauts

Ninhydrin

Molisch

(-/+)

Conclusion

Through trituration with different solvents,

various types of lipids in a calfs brain were
extracted. Cholesterols lack of phosphorylation
and polarity were observed when it was isolated
and dissolved via acetone.
In addition, hexanes properties allowed it
to be isolated through solvating with hexane.
Lastly, hot ethanol, a specific solvent for
sphingosine was used to isolate sphingosine.
In all of the procedures used in isolating
the various lipids, methanol:chloroform mixture
was used for its preferrable properties as a
solvent.

Based from the experiment, it can be

inferred that the extracted cholesterol and standard
cholesterol produced positive results in the
Liebermann-burchard test and Salkowski test due
to the presence of sterols.

Furthermore, it can be inferred that both

lecithin and glycerophosphatides yielded positive
results in the phosphate test due to the presence of
phosphate groups.

In addition, lecithin tested positive in

the krauts and Ninhydrin test due to
the presence of choline as well as
free amino groups.
Lastly, only galactocerebroside
produced a positive result in the
Molisch test due to the presence of a
carbohydrate in its head group.

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WB Saunders Company.
Chawla, R. (2003). Practical Clinical Biochemistry: Methods and Interpretations. India:
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Christie, W. (1993). Advances in Lipid Methodology- Two. Dundee: Oily Press. Clark, J.
(1964). Experimental Biochemistry. San Francisco: WH Freeman and Company.
Domodoran, G. (2011). Practical Biochemistry. New Delhi: Jaypee Brothers Medical
Publisher (P) Ltd.
Krishnaswamy, N.R. (2003). Chemistry of Natural Products: A Laboratory Handbook.
India: Universities Press Private Ltd.
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http://www.wiredchemist.com/chemistry/instructional/laboratory-tutorials/recrystallization